Cowpea genome sequence (cultivar IT86D-1010)

Full description

This resource is a de novo genome assembly of the cowpea (Vigna unguiculata (L.) Walp) cultivar IT86D-1010. This cultivar has become the preferred germplasm for developing transgenic cowpea plants, either genetically modified or gene edited. For the production and characterization of genetically modified cowpeas, it is crucial to have a high-quality genomic sequence of the cultivar being transformed.
Although there are publicly accessible genomic sequences for cowpea, the reference cultivar is IT97K-499-35 (Lonardi et al., 2019). There is also a previously published version of IT86D-1010 based on Illumina short reads (Spriggs, et al, 2018), however this is too fragmented.
The submitted dataset is a new, high-quality, assembled genomic sequence for the cowpea cultivar IT86D-1010, using Oxford Nanopore Technology (ONT) long read sequencing, and corrected using the already published Illumina short reads (Spriggs, et al., 2018). The resulting genome consisted of 505 contigs, with a total assembly length of 537341206, and was comparable in quality to the IT97K-499-35 reference.

Lineage: Plant material and tissue sample

Cowpea (Vigna unguiculata) cultivar IT86D-1010 was originally sourced from the International Institute of Tropical Agriculture (IITA). Line have been maintained in CSIRO for more than 10 generations (not through single seed descent). Young unexpanded leaves were collected for DNA extraction.

DNA Isolation and Sequencing

For Illumina short read sequencing, DNA extraction was carried out using a Qiagen maxi DNA kit as per the manufacturer’s instructions. Illumina library preparation and sequencing of DNA was undertaken by the Australian Genome Research Facility (AGRF) with 2 × 100 bp standard insert paired-end sequencing using a Hiseq 2500 system, as described in Spriggs, et al. (2018),

For Oxford Nanopore Technology (ONT) long read sequencing, DNA was isolated using the QIAGEN Genomic-tip as per the manufacturer’s instructions. DNA was extracted from a transformed IT86D-1010 line. Shorter DNA fragments (<5 kb) were depleted from the DNA sample using the Circulomics SRE XS protocol. The size-selected genomic DNA was then prepared for sequencing using the ONT native barcoding workflow and then sequenced on an ONT PromethION Sequencer at the Biomolecular Resource Facility (BRF) at the John Curtin School of Medical Research, Australian National University (Canberra, ACT).

Genome sequencing

Adapter sequences of the short read sequencing data were removed from the resulting reads using TrimGalore v0.6.6. The long-read sequences were trimmed using Porechop v0.2.4. Long-reads were assembled using Flye v4.0.8 under default settings. The resulting genome assembly was then subjected to three rounds of polishing using long reads and three rounds using short reads with Racon v1.4.22 under default settings. This was followed by six rounds of short-read-based polishing using Polca MaSURCA v4.0.7 using default settings. The resulting genome sequence was sequentially decontaminated using The NCBI Foreign Contamination Screen (FCS) tool (fcs-adaptor and fcs-gx) to obtain the final contig assembly. The sequences corresponding to the T-DNA insertion were identified and removed. The assembly was assessed using BUSCO v5.2.2 against the lepidoptera (lepidotptera_odb10) and Insecta (insecta_odb10) lineages’ gene sets.

Genome annotation

The genome was annotated with Liftoff (V16.3) using the published cowpea cultivar IT97K-499-35 (GCF_004118075.2) as a reference (Lonardi et al., 2019).


References:
Spriggs A, Henderson ST, Hand ML, Johnson SD, Taylor JM, Koltunow A. Assembled genomic and tissue-specific transcriptomic data resources for two genetically distinct lines of Cowpea ( Vigna unguiculata (L.) Walp). Gates Open Res. 2018, 18;2:7. doi: 10.12688/gatesopenres.12777.2.
Lonardi S, Muñoz-Amatriaín M, Liang Q, Shu S, Wanamaker SI, Lo S, Tanskanen J, Schulman AH, Zhu T, Luo MC, Alhakami H, Ounit R, Hasan AM, Verdier J, Roberts PA, Santos JRP, Ndeve A, Doležel J, Vrána J, Hokin SA, Farmer AD, Cannon SB, Close TJ. The genome of cowpea (Vigna unguiculata [L.] Walp.). Plant J. 2019, 98:767-782. doi: 10.1111/tpj.14349.